Human erythroid p55 of the present invention was originally identified as a 55 kDa phosphoprotein that copurified with preparations of dematin, an actin-bundling protein of human erythrocyte membranes (1-3). Although fractions enriched with the 55 kDa protein completely inhibited dematin's actin-bundling activity, this inhibitory activity was traced to the presence of a protein kinase distinct from the 55 kDa protein (1-3).
Lacking any protein kinase function, independent interaction with actin, or any other known function, the 55 kDa protein was no longer of interest with respect to actin-bundling activity in erythrocytes. However, the subsequent molecular cloning and sequencing of human erythroid p55 in accordance with the present invention revealed the unexpected presence of the src homology 3 (SH3) motif conserved in regulatory domains of the non-receptor class of oncogene-encoded tyrosine kinases (4-6, 43-44). Further characterization of human erythroid p55 in accordance with the invention surprisingly revealed that the C-terminal half of p55 also contains an enzymatically active guanylate kinase domain with significant homology to guanylate kinases of yeast and E. coli.
The importance of the SH3 motif is underscored by mutational studies which indicate that the SH3 motif may play a role in the suppression of tyrosine kinase activity in vivo and may participate in the process of tumor suppression (14-16). For example, mutations in the N-terminal SH3-containing regions of the srs and abl gene products are known to activate their oncogenic potential (14,15). The neuronal src gene product, which has enhanced tyrosine kinase activity, contains a hexapeptide insert in the SH3 motif (16). Suppressive activity of the SH3 sequence is further evidenced by the inhibition of tyrosine kinase activity of the v-src product in vitro by a synthetic peptide corresponding to part of the SH3 motif (17).
The presence of the SH3 motif in a diverse group of cytoskeletal and signal-transducing proteins unrelated to tyrosine kinase supports the hypothesis that it may mediate binding to regulatory ligand(s) common to these proteins (6). Such a ligand may block the transforming ability of various oncogene-encoded tyrosine kinases (15). Alternatively, the SH3 motif may mediate recognition of substrate molecules and regulate their proximity to the plasma membrane.
The amino acid sequence of p55, including both SH3 and guanylate kinase domains, as well as an N-terminal undefined domain, is also very similar to the primary sequence of the Drosophila tumor suppressor gene product dlg. It was recently demonstrated that recessive lethal mutations of the Drosophila dlg locus produced neoplastic overgrowth in imaginal discs (45). More specifically, such mutations caused a loss in apical-basal cell polarity and induced proliferation of epithelial tissues (45). These studies predict that the guanylate kinase domain of p55 may influence signal transduction and tissue proliferation by modulating guanine nucleotide levels at the plasma membrane (45).
The sequencing and characterization of human erythroid p55, and its use in research and clinical applications, thus provide various aspects of the present invention described more fully below.